The use of acridinium esters as chemiluminescent labels in clinical assays is known. For example, European Patent Application No. 82 306 557.8 describes the use of an aryl acridinium ester activated with an N-hydroxy-succinimidyl moiety as a chemiluminescent label in immunoassays. U.S. Pat. Nos. 4,745,181; 4,918,192; 5,241,070, and Copending U.S. patent application Ser. Nos. 08/032,947 filed Jan. 26, 1994, and 08/032,085 filed Mar. 17, 1993, describe polysubstituted aryl acridinium esters (PAAE) which are useful in immunoassays and nucleic acid hybridization assays. U.S. Pat. No. 5,227,489 and Copending divisional U.S. patent application Ser. No. 08/032,231 filed Mar. 17, 1993 which is the parent to the instant application, describe hydrophilic polysubstituted aryl acridinium esters and lumisome conjugates thereof useful in clinical assays, particularly those assays involving liposomes.
Previous methods for the synthesis of 2',6'-Dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl-10-Methyl-9-acridineca rboxylate Methylsulfate (DMAE-NHS) as described in U.S. Pat. No. 4,745,181, require the use of a phenoxy group substituted with a benzyloxycarbonyl group as an intermediate to form the acridine ester via a long synthetic pathway. It is desirable to develop new and efficient methods of synthesizing the useful acridinium ester labels of the instant invention. The unexpected ability to form 2'6'-dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl 9-acridinecarboxylate (DMAeE-NHS) by the simplified procedure of combining a solution of succinimidyl 3,5-dimethyl-4-hydroxybenzoate and 4-dimethylaminopyridine with 9-acridinecarbonyl chloride hydrochloride, was not readily predictable in view of the coexistence of two reactive leaving groups in the same reaction, an acid chloride from 9-acridinecarbonyl chloride and a succinimidyl ester from succinimidyl 3,5-dimethyl-4hydroxybenzoate. For example, in the case where N-succinimidyl 3(4-hydroxyphenyl)-propionate is reacted with the 9-acridinecarbonyl chloride referred to above, the condensation can be carried out under mild conditions (approximately room temperature) due to the absence of the two methyl groups in the ortho positions. (See for example U.S. Pat. No. 4,946,958, cols. 4-5, where the reactant does not contain the ortho-substituted methyl groups.) On the other hand, when the two methyl groups are present, much more drastic conditions (100 degrees C. for 2 hours) are required for the condensation reaction to take place, due to the steric hindrance caused by the methyl groups. It should be noted, though, that there is a benefit provided by the presence of the methyl groups, namely the added stability of the resulting acridinium ester, as discussed, for example, in U.S. Pat. No. 4,745,181.
The lipophilic nature of the prior art acridinium esters and other chemiluminescent compounds render them unsuitable for encapsulation within liposomes because of their rapid leakage through the liposome wall. Additionally, the limited water solubility of prior art acridinium esters and other chemiluminescent compounds only allow the encapsulation of a few marker molecules per liposome vesicle, resulting in relatively low signal amplification.
The novel functionalized hydrophilic acridinium esters of the instant invention are useful in immunoassay, yield results that are superior in sensitivity to prior methods, and do not need to use dangerous radiolabels, or organic enzyme/substrates. The use of chemiluminesent labels of this type resulted an unexpected improvement over prior methods and can lead to the functional improvement of previously inoperable or inaccurate assay methodologies. The novel discovery that hydrophilic acridinium esters of the instant invention could be used to label biomolecules and compounds directly without reducing the solubility of the complex allows for many applications in the field of immunoassay. The novel compounds of the instant invention will allow for sensitive immunoassay without the need of excessive use of reagents in the reaction mixture, and are thus designed to reduce non-specific interactions, while not interfering with desired specific interactions.
Another unexpected benefit of having an PAAE carrying a hydrophilic moiety, particularly as a substituent at the nitrogen atom of the acridinium nucleus, is the significant improvement of the chemiluminescent quantum yield relative to that of acridinium esters which simply have an alkyl group (U.S. Pat. No. 4,745,181) or carboxymethyl group (G. Zomer et al., 1989, Anal. Chem. Acta 227: 11-19) substituted at the same position.
The area of immunoassay is well developed, and it is the discovery of unique labeling systems that can spur a quantum advancement in the field of immunoassay. In the field of immunoassay it is desirable to have assays which are highly specific, and highly sensitive to low concentrations, and yet still yield detectable and distinguishable signals for measurement. The instant invention allows for the specific detection of analytes at low concentration, without loss of solubility. The instant invention provides a means for making many specific labeling agents which can be used to detect low levels of analytes. The instant invention also teaches one with ordinary skill in the art, a novel and useful means of performing immunoassay that lends itself readily to automation and commercialization as assay kits and reagent kits.
The application of the novel acridinium esters of the instant invention in conjugates with bioactive proteins such as avidin, antibodies, DNA binding proteins, histones, and ribosomes, and others, is made possible by the hydrophilic properties of the synthesized compounds. These novel acridinium esters are also useful for the labeling of isolated, or intact, RNA, DNA, proteins, peptides, inactivated protein, neurotransmitters, hormones, viruses, viral antigens, bacteria, bacterial antigens, toxins, cytokines, antibody fragments, receptor proteins and other such targets both in vitro, and in vivo. The sampling of tissue samples, serum samples or other such biological samples, and the detection of previously difficult to measure specific analytes in rapid assays, are made possible by the compositions and methods of the instant invention.
One specific application of the instant invention is in the field of pathogen detection. While there have been many assays developed to detect viral pathogens, such as Rubella, the instant invention provides a unique and highly sensitive assay which is unexpectedly superior to previous chemiluminescent methods. The instant methods are comparable with conventional assays in sensitivity yet are more efficient in time required to perform the assay.
Another application of the instant invention is in the area of detection of hormones or haptens or other small biologically active molecules in biological samples. Where the hormone or hapten levels are low, and transient, it is useful to have a sensitive and rapid method of testing for, and measuring the levels of, hormones or haptens in such biological samples. One of the possible examples is the detection of steroid hormone levels such as testosterone. Detection of testosterone using homologous and heterologous hapten conjugates is known. In this methodology, antibodies are generated to one form of testosterone immunogen. Then said antibodies are used to detect testosterone in a sample, while a conjugated competitive hapten tracer, that is of a different form than the immunogen, is added. Homologous assays are burdened with unacceptably high cross-reactivity such as when using C4-testosterone-B-gal conjugate haptens, and T-3-O-CMO-glucoamylase conjugates. Somewhat lower crossreactivity has been obtained with 11a-substituted hapten-HRP, and T-3-O-CMO hapten-penicillinase (Rao et al., 1992, Steroids 57: 154-162). The instant invention provides for superior detection over prior heterologous hapten assay in that the hydrophilic acridinium ester labels of the instant invention contribute to reduced non-specific interactions.
Accordingly, it is the purpose of the instant invention to provide novel functionalized hydrophilic acridinium esters and conjugates thereof for use as chemiluminescent tracers. It is also a purpose of the instant invention to provide for novel methods of detecting an analyte using functionalized hydrophilic acridinium esters and conjugates thereof. It is also a purpose of the instant invention to provide a new improved synthetic procedure for the efficient production of acridine esters.